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pSBCAR1 ROR1 (4A5) h(28OXζ) (CAR-SB-02LX399)

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All products and services are For Research Use Only and CANNOT be used in the treatment or diagnosis of disease.

Sleeping Beauty (SB) transposon, a type of nonviral integrative vectors, provides an alternative to modify primary T cells. Creative biolabs has developed SB transposon CAR vector pSBCAR1 ROR1 (4A5) h(28OXζ), which is constructed for the engineering of T cells to target human ROR1. The T cells are genetically modified through transduction with a nonviral vector expressing scFv of anti-ROR1 antibody linked to a CD28 transmembrane domain/ endodomain and OX40, CD3-zeta signaling domains. And the vector product was designed for the treatment of Cancer.

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Details

  • Target
  • ROR1
  • Targeting Cell Type
  • T cell
  • Targeting Diseases
  • Cancer
  • Generation
  • Third
  • Vector Name
  • pSBCAR1
  • Vector Length
  • ~6kb
  • Vector Type
  • Sleeping Beauty (SB) transposon
  • Receptor Construction
  • scFv-CD28-OX40-CD3ζ
  • Discription of Signaling Cassetes
  • CD28
    CD28 (Cluster of Differentiation 28) is one of the proteins expressed on T cells that provide co-stimulatorysignals required for T cell activation and survival. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins which are expressed on antigen-presenting cells(APC). CD28 modulates the primary TCR/CD3ζ signal in a different fashion than the late costimulatory elements OX40 and 4-1BB. CD28 enhances the expression of downstream regulators that impact on T-cell proliferation, death, differentiation, and effector functions. CAR+ T cells containing the CD28 endodomain showed strikingly enhanced sustained T cell activation, growth, survival. And CD28 results in a brightly expressed, stable receptor as the transmembrane domain. Including CD28 costimulatory domains in CARs led to enhanced anti-malignancy efficacy.
    OX40
    OX40, also known as CD134 or TNFRSF4 is a member of the TNFR-superfamily of receptors which is not constitutively expressed on resting naïve T cells, unlike CD28. OX40 is a secondary co-stimulatory immune checkpoint molecule, expressed after 24 to 72 hours following activation. The interaction between OX40 and its binding partner, OX40L (CD252) plays an important role in antigen-specific T-cell expansion and survival. OX40 and OX40L also regulate cytokine production from T cells and modulate cytokine receptor signaling. OX40 cosignaling in CAR improve redirected T-cell effector functions and enhance anti-tumor activity.
    CD3ζ
    CD3ζ, also known as T-cell receptor zeta, which together with T-cell receptor and CD3γ, δ , ε chain, forms the TCR-CD3 complex. ζ was expressed independently from the complex. The zeta chain plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. CD3-zeta,which contains 3 ITAMs, is the most commonly used endodomain component of CARs. It transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signaling is needed. For example, chimeric CD28 and OX40 can be used with CD3-zeta to transmit a proliferative/survival signal, or all three can be used together.

Target

  • Clone
  • 4A5
  • Host
  • Humanized
  • Target Species
  • Human
  • Gene Name
  • Receptor tyrosine kinase like orphan receptor 1
  • Synonyms
  • ROR1; NTRKR1; dJ537F10.1

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  • Published Data
CAR scFv data FCM

Fig.1 MEC1 or MEC1-ROR1 cells were collected from the marrow or spleens of mice engrafted 3 weeks earlier with MEC1 or MEC1-ROR1 cells. The fluorescence of cells stained with 4A5–Alexa Fluor 647 (ordinate) and anti-CD19-PE (abscissa) are shown in the contour
plots.

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Fig.1 MEC1 or MEC1-ROR1 cells were collected from the marrow or spleens of mice engrafted 3 weeks earlier with MEC1 or MEC1-ROR1 cells. The fluorescence of cells stained with 4A5–Alexa Fluor 647 (ordinate) and anti-CD19-PE (abscissa) are shown in the contour plots.

The percentages at the top right of each contour plot indicate the proportions of cells with fluorescence above the
threshold indicated by the dotted line.

Yu, J., Chen, L., Cui, B., Widhopf, G. F., Shen, Z., Wu, R., ... & Kipps, T. J. (2016). Wnt5a induces ROR1/ROR2 heterooligomerization to enhance leukemia chemotaxis and proliferation. The Journal of clinical investigation, 126(2), 585-598.

CAR scFv data FCM

Fig.2 Flow cytometric analysis of representative 7-mo-old ROR1×TCL1 (Left), TCL1(Center), or ROR1 (Right) Tg mice depicting theexpansion of CD5+B220lowB cells in the blood.

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Fig.2 Flow cytometric analysis of representative 7-mo-old ROR1×TCL1 (Left), TCL1(Center), or ROR1 (Right) Tg mice depicting theexpansion of CD5+B220lowB cells in the blood.

ROR1 interacts with TCL1 and acceleratesCD5+B-cell leukemogenesis in TCL1 Tg mice.

Widhopf, G. F., Cui, B., Ghia, E. M., Chen, L., Messer, K., Shen, Z., ... & Kipps, T. J. (2014). ROR1 can interact with TCL1 and enhance leukemogenesis in Eµ-TCL1 transgenic mice. Proceedings of the National Academy of Sciences, 111(2), 793-798.

CAR scFv data FCM

Fig.3 Flow-cytometric analysis of OV1110, AA1581, or AA0857. Thecells were stained with 4A5 or control mAb, and with ALDOFLUOR without(−) or with (+) the ALDH1 inhibitor DEAB, as indicated at the top.

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Fig.3 Flow-cytometric analysis of OV1110, AA1581, or AA0857. Thecells were stained with 4A5 or control mAb, and with ALDOFLUOR without(−) or with (+) the ALDH1 inhibitor DEAB, as indicated at the top.

The open boxes in the each contour plot indicate the gates for identifying cells withALDH1 activity, the proportion of which is indicated

Zhang, S., Cui, B., Lai, H., Liu, G., Ghia, E. M., Widhopf, G. F., ... & Kipps, T. J. (2014). Ovarian cancer stem cells express ROR1, which can be targeted for anti–cancer-stem-cell therapy. Proceedings of the National Academy of Sciences, 111(48), 17266-17271.

CAR scFv data FCM

Fig.4 Human cancer cell lines that expressed the ROR1 antigen.

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Fig.4 Human cancer cell lines that expressed the ROR1 antigen.

Jeko-1 were stained with anti-ROR1 antibody (clone 4A5) or isotype control and analyzed by flow cytometry.

Lee, B. K., Wan, Y., Chin, Z. L., Deng, L., Deng, M., Leung, T. M., ... & Zhang, H. (2022). Developing ROR1 Targeting CAR-T Cells against Solid Tumors in Preclinical Studies. Cancers, 14(15), 3618.

CAR scFv data FuncS

Fig.1 Competition assay using anti-ROR1 monoclonal antibody (clone 4A5).

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Fig.1 Competition assay using anti-ROR1 monoclonal antibody (clone 4A5).

Histograms show the binding of anti-ROR1 4A5 monoclonal antibodies to cell surface ROR1 protein.

Daikuzono, H., Yamazaki, M., Sato, Y., Takahashi, T., & Yamagata, K. (2021). Development of a DELFIA method to detect oncofetal antigen ROR1-positive exosomes. Biochemical and Biophysical Research Communications, 578, 170-176.

CAR scFv data ELISA

Fig.2 Binding specificity and affinity of D10 and 4A5 mAb for ROR1

CAR Construction : Latest CAR Construction

Fig.2 Binding specificity and affinity of D10 and 4A5 mAb for ROR1

Affinity measurements of binding of the D10 and 4A5 mAb to recombinant ROR1 protein using a KinExa 3200 instrument.

Widhopf, G. F., Cui, B., Ghia, E. M., Chen, L., Messer, K., Shen, Z., ... & Kipps, T. J. (2014). ROR1 can interact with TCL1 and enhance leukemogenesis in Eµ-TCL1 transgenic mice. Proceedings of the National Academy of Sciences, 111(2), 793-798.

CAR scFv data FCM

Fig.3 The D10 and 4A5 mAb bind to non–cross-blocking epitopes of human ROR1.

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Fig.3 The D10 and 4A5 mAb bind to non–cross-blocking epitopes of human ROR1.

Fluorescence histograms of the EW36 human B-cell line stained with Alexa-647-conjugated 4A5 (Upper) or Alexa-647-conjugated D10 (Lower) mAb.

Widhopf, G. F., Cui, B., Ghia, E. M., Chen, L., Messer, K., Shen, Z., ... & Kipps, T. J. (2014). ROR1 can interact with TCL1 and enhance leukemogenesis in Eµ-TCL1 transgenic mice. Proceedings of the National Academy of Sciences, 111(2), 793-798.

More Published Data More Published Data

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For research use only. Not intended for any clinical use. No products from Creative Biolabs may be resold, modified for resale or used to manufacture commercial products without prior written approval from Creative Biolabs.

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